The estrogen receptor (ER) is a master regulator and driver of 70% of breast cancers, making the ER a primary therapeutic target. Hypoxia, common in ER+ breast tumours, plays a central role in tumour development and response to treatment. Hypoxia inducible factor-1-alpha (HIF-1α) is a transcription factor which when stabilised by dimethyloxalylycine (DMOG), robustly reprogrammed oncogenic ER binding, a preliminary finding by the Holding lab via ChIP-seq analysis.
Building on this ChIP-seq analysis, this project aimed to investigate GREB1, PS2 and CASC4 protein alterations in response to DMOG by western blot analysis. Then to investigate this on a transcriptional level, we performed RT-qPCR of GREB1, PS2 and CASC4. Using proximal ligation assay (PLA), we aimed to confirm that HIF-1α and ERα are within proximity (40nm), to suggest a protein-protein interaction. All assays were performed using MCF-7 and T47-D ER+ breast cancer cell lines. The hypothesis was that loss and/or gain in ER binding in response to HIF-1α stabilisation will result in an increase or decrease respectively of the nearby gene’s transcripts and protein products based on changes in ER binding.
Western blot analysis showed a potential increase in GREB1 protein in each cell line. In addition, an increase in PS2 protein in MCF-7 cell lines. These results did not support the initial hypothesis. Changes in CASC4 protein were inconclusive. RT-qPCR analysis of GREB1, PS2 and CASC4 show that mRNA alterations are cell line-specific in response to 2mM DMOG. PS2 mRNA was decreased upon HIF-1α stabilisation in both cell lines, and CASC4 mRNA was increased on HIF-1α activation, both results supporting the initial hypothesis. PLA data was inconclusive, Optimisation conditions of each assay established in this project provides the grounding for continuation of assays to establish additional data for more reliable results.
